OPTIMIZATION OF ONCORETROVIRAL VECTORS ENCODING RNA DECOYS

编码 RNA 诱饵的肿瘤逆转录病毒载体的优化

• 批准号: 7349504
• 负责人: Stephen E. Braun
• 金额: $ 13.48万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7349504
• 关键词: OPTIMIZATION; ONCORETROVIRAL; VECTORS; ENCODING; RNA; OPTIMIZATION; ONCORETROVIRAL; VECTORS; ENCODING; RNA

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. RNA decoys have a number of advantages for the inhibition of HIV replication, including their lack of immunogenicity and their ability to target conserved genes essential for viral replication. However, optimal inhibition of viral replication by RNA decoys has generally been obtained with multimeric RNA decoys, which significantly increase the risk of transgene instability when delivered using retroviral vectors. We therefore examined a number of parameters affecting the ability of oncoretroviral vectors to stably deliver HIV-1 RNA decoys and inhibit viral replication. For the retroviral backbone, we chose the oncoretroviral vector MMP, which does not contain a selectable marker gene, and generated a series of vectors with and without intact splice donor and splice acceptor signals, and with the oncoretroviral LTR or an internal HIV-1 LTR transcriptionally regulating the polymeric TAR and RRE RNA decoys. By decreasing the number of TAR decoys, vector stability was increased, resulting in more efficient inhibition of viral replication in transduced cells. Inclusion of an internal HIV promoter within the retroviral vector provided more consistent viral inhibition. The efficiency of these different vectors has been evaluated in multiple T cell clones derived from transduced cells and stable high titer producer cell lines generated. Transduction of autologous CD34+ bone marrow cells with one of these vectors has resulted in stable gene marking of 0.1 percent - 1 percent of lymphocytes. These optimized vectors will facilitate analysis of the efficacy of RNA decoys for stem cell gene for AIDS in a nonhuman primate model.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。 RNA诱饵在抑制HIV复制方面具有许多优势，包括它们缺乏免疫原性以及靶向病毒复制必不可少的保守基因的能力。但是，通常用多聚体RNA诱饵获得了对RNA诱饵对病毒复制的最佳抑制作用，当使用逆转录病毒载体传递时，这会显着增加转基因不稳定性的风险。因此，我们检查了许多参数，这些参数影响了癌逆转录病毒载体稳定传递HIV-1 RNA诱饵并抑制病毒复制的能力。 For the retroviral backbone, we chose the oncoretroviral vector MMP, which does not contain a selectable marker gene, and generated a series of vectors with and without intact splice donor and splice acceptor signals, and with the oncoretroviral LTR or an internal HIV-1 LTR transcriptionally regulating the polymeric TAR and RRE RNA decoys.通过减少焦油诱饵的数量，载体稳定性增加，从而更有效地抑制了转导细胞中病毒复制。在逆转录病毒载体中纳入内部HIV启动子提供了更一致的病毒抑制作用。这些不同载体的效率已经在源自转导的细胞和产生的稳定的高滴度产生细胞系的多个T细胞克隆中进行了评估。自体CD34+骨髓细胞中具有其中一种载体的转导导致稳定的基因标记为0.1％ - 1％的淋巴细胞。这些优化的载体将促进分析非人类灵长类动物模型中RNA诱饵对干细胞基因的辅助工具的功效。